Mosquito feeding patterns in the context of West Nile and eastern equine encephalitis viruses in eastern Ontario, Canada.

Eastern equine encephalitis (EEE) virus is a highly pathogenic mosquito-borne zoonosis that is responsible for outbreaks of severe disease in humans and equines, resulting in high mortality or severe neurological impairment in most survivors. In the northeastern United States, EEE virus is maintained in an enzootic cycle involving the ornithophilic mosquito, Culiseta melanura (Coquillett) and passerine birds in freshwater swamp habitats. To evaluate the role of Cs. melanura and Culiseta morsitans (Theobald) in recent episodes of EEE virus activity in Massachusetts, we collected blood-fed mosquitoes between June, 2007, and October, 2008, from virus foci in 6 counties, and identified the source of blood meals by PCR amplification of mitochondrial cytochrome b gene and sequencing. Analysis of 529 Cs. melanura and 25 Cs. morsitans revealed that nearly 99% and 96% of mosquitoes, respectively, acquired blood meals solely from avian hosts. American Robin, Turdus migratorius Linnaeus was identified as the most common vertebrate host for Cs. melanura (21.7%, n=115), followed by Tufted Titmouse, Baeolophus bicolor (L.) (8.7%, n=46), Black-capped Chickadee, Poecile atricapillus (L.) (8.5%, n=45), Scarlet Tanager, Piranga olivacea (Gmelin) (6.8%, n=36), Field Sparrow, Spizella pusilla (Wilson) (6.2%, n=33), Northern Cardinal, Cardinalis cardinalis (L.) (5.7%, n=30), and other mostly Passeriformes birds. Mammalian-derived blood meals were identified as white-tailed deer, Odocoileus virginianus Zimmermann, domestic cow, Bos taurus L., and human, Homo sapiens L. There were 4 isolations of EEE virus, West Nile virus, and Highland J virus from Cs. melanura. Our results in conjunction with other lines of evidence, including reservoir competency, prevalence of antibody, and infection in nature, suggest that the American Robin, Tufted Titmouse, Black-capped Chickadee, and a few other passerine birds may play key roles in supporting EEE virus transmission in Massachusetts. Infrequent blood feeding of Cs. melanura on mammalian hosts, including humans, also indicates that this mosquito may occasionally contribute to epidemic/epizootic transmission of EEE virus in this region.

BackgroundEastern equine encephalitis (EEE) virus (Togaviridae, Alphavirus) is a highly pathogenic mosquito-borne zoonosis that is responsible for occasional outbreaks of severe disease in humans and equines, resulting in high mortality and neurological impairment in most survivors. In the past, human disease outbreaks in the northeastern U.S. have occurred intermittently with no apparent pattern; however, during the last decade we have witnessed recurring annual emergence where EEE virus activity had been historically rare, and expansion into northern New England where the virus had been previously unknown. In the northeastern U.S., EEE virus is maintained in an enzootic cycle involving the ornithophagic mosquito, Culiseta melanura, and wild passerine (perching) birds in freshwater hardwood swamps. However, the identity of key avian species that serve as principal virus reservoir and amplification hosts has not been established. The efficiency with which pathogen transmission occurs within an avian community is largely determined by the relative reservoir competence of each species and by ecological factors that influence contact rates between these avian hosts and mosquito vectors.Methodology and principle findingsContacts between vector mosquitoes and potential avian hosts may be directly quantified by analyzing the blood meal contents of field-collected specimens. We used PCR-based molecular methods and direct sequencing of the mitochondrial cytochrome b gene for profiling of blood meals in Cs. melanura, in an effort to quantify its feeding behavior on specific vertebrate hosts, and to infer epidemiologic implications in four historic EEE virus foci in the northeastern U.S. Avian point count surveys were conducted to determine spatiotemporal host community composition. Of 1,127 blood meals successfully identified to species level, >99% of blood meals were from 65 avian hosts in 27 families and 11 orders, and only seven were from mammalian hosts representing three species. We developed an empirically informed mathematical model for EEE virus transmission using Cs. melanura abundance and preferred and non-preferred avian hosts. To our knowledge this is the first mathematical model for EEE virus, a pathogen with many potential hosts, in the northeastern U.S. We measured strong feeding preferences for a number of avian species based on the proportion of mosquito blood meals identified from these bird species in relation to their observed frequencies. These included: American Robin, Tufted Titmouse, Common Grackle, Wood Thrush, Chipping Sparrow, Black-capped Chickadee, Northern Cardinal, and Warbling Vireo. We found that these bird species, most notably Wood Thrush, play a dominant role in supporting EEE virus amplification. It is also noteworthy that the competence of some of the aforementioned avian species for EEE virus has not been established. Our findings indicate that heterogeneity induced by mosquito host preference, is a key mediator of the epizootic transmission of vector-borne pathogens.Conclusion and significanceDetailed knowledge of the vector-host interactions of mosquito populations in nature is essential for evaluating their vectorial capacity and for assessing the role of individual vertebrates as reservoir hosts involved in the maintenance and amplification of zoonotic agents of human diseases. Our study clarifies the host associations of Cs. melanura in four EEE virus foci in the northeastern U.S., identifies vector host preferences as the most important transmission parameter, and quantifies the contribution of preference-induced contact heterogeneity to enzootic transmission. Our study identifies Wood Thrush, American Robin and a few avian species that may serve as superspreaders of EEE virus. Our study elucidates spatiotemporal host species utilization by Cs. melanura in relation to avian host community. This research provides a basis to better understand the involvement of Cs. melanura and avian hosts in the transmission and ecology of EEE virus and the risk of human infection in virus foci.

A traditional nested reverse transcription-polymerase chain reaction (RT-PCR) assay specific for eastern equine encephalomyelitis (EEE) virus was designed to multiplex with a previously described West Nile (WN) virus nested RT-PCR assay. Differentiation of EEE and WN was based on base pair size of the amplified product. One hundred fifty-seven mammalian and avian brain tissues were tested by EEE/WN nested multiplex RT-PCR, EEE nested RT-PCR, and WN nested RT-PCR, and results were compared with other diagnostic test results from the same animals. Serological and virus isolation testing confirmed the results of the multiplex PCR assay. When compared with cell culture virus isolation, the multiplex assay was shown to be more sensitive in detecting the presence of EEE or WN virus in brain tissues. The multiplex assay was shown to be sensitive and specific for North American EEE and WN and provided a rapid means of identifying both viruses in brain tissues. No apparent sacrifice in sensitivity was observed in the multiplex procedure compared with the individual EEE and WN nested RT-PCR assays. Data collected from an additional 485 multiplex RT-PCR tests conducted during the summer and fall of 2002 further support the validity of the procedure.

West Nile Virus Disease and Other Arboviral Diseases—United States, 2011

During August and September of 1991, an epizootic of eastern equine encephalitis (EEE) virus in horses occurred in Wayne and Holmes countries, OH. This was the first recorded epizootic of EEE virus in the state. Twelve horses were confirmed positive for EEE virus through virus isolation or seroconversion, and seven additional horses with compatible symptoms were in close spatial and temporal proximity to the confirmed cases and were presumed to have died from EEE virus. The outbreak was centered around the Killbuck Wildlife Area, a 2,147-ha tract maintained by the state, half of which consists of wooded swamp and marsh. Mosquitoes were collected in upland areas before the epizootic and in the swamp basin at the end of the epizootic to identify the mosquito species involved in EEE virus transmission. We collected and tested 22,095 specimens for the presence of virus. EEE virus was isolated from one pool of the most likely epizootic vector, Coquillettidia perturbans (Walker). The minimum infection rate for EEE virus in this species was 0.1/1,000. Dense populations of Aedes vexans (Meigen) and Culex salinarius Coquillett occurred in the area, but their densities peaked after the epizootic. It is unlikely that these species were involved in epizootic transmission. IgM antibody to EEE virus was detected in three bird species collected in the swamp.

Immunohistochemistry (IHC) and in situ hybridization (ISH) can be used either to detect or to differentiate between Eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) within formalin-fixed, paraffin-embedded (FFPE) brain tissue of horses. To compare the diagnostic sensitivity and specificity of ISH and IHC, FFPE brain tissue from 20 EEEV-positive horses and 16 WNV-positive horses were tested with both EEEV and WNV oligoprobes and EEEV- and WNV-specific antibodies. Reverse transcription polymerase chain reaction (RT-PCR) for detection of EEEV and WNV was used as the gold standard to confirm infection. All horses that tested positive for EEEV by RT-PCR also tested positive by IHC and ISH, except for 1 case that was false-negative by ISH. In contrast, all horses that tested positive for WNV by RT-PCR tested negative by IHC and only 2 horses tested positive by ISH. No false-positives were detected with either method for both viruses. Both IHC and ISH are highly specific and sensitive diagnostic methods to detect EEEV in equine FFPE brain tissues, although neither appear effective for the diagnosis of WNV in equine neurologic cases.

Surveillance of Encephalitis-Causing Arboviruses in Horses in South Korea

Toward a more detailed understanding of the Eastern equine encephalitis virus (EEEV) cycle, individual blood-fed Culiseta melanura (Coquillett) mosquitoes were collected at an enzootic focus in New York State. Blooded females were tested for EEEV by multiplex real-time quantitative reverse transcription polymerase chain reaction, qRT-PCR. Host bloodmeals were identified using DNA amplification and sequencing of the cytochrome B gene. In 2018, 577 individual mosquitoes were tested. Virus was detected in the bodies of 16, of which two had the virus in their legs. In 2022, 606 individual mosquitoes were tested, and the virus was detected in the bodies of six, of which one had the virus in its legs. Virus in the legs suggested a disseminated infection. The qRT-PCR cycle threshold (Ct) values for individual bodies ranged from 17.6 to 38.3, and for legs, 22.8 to 28.1. Host sources for 17 of the 22 were: Passeriformes, Bombycillidae, cedar waxwings (Bombycilla cedrorum); Icteridae, red-winged blackbird (Agelaius phoeniceus); Turdidae, American robin (Turdus migratorius), veery (Catharus fuscescens), and wood thrush (Hylocichla mustelina); Vireonidae, yellow-throated vireo (Vireo flavifrons); Passerellidae, song sparrow (Melospiza melodia), and field sparrow (Spizella pusilla); Parulidae, American redstart (Setophaga ruticilla); and Gruiformes, Rallidae, American coot (Fulica americana). In both years, red-winged blackbirds were a source of blood for Cs. melanura in the earliest 2 d after EEEV was detected. Annually, the earliest detection of the virus was in blooded-gravid mosquitoes before non-blooded mosquitoes in 15 of 17 yr. These results support the thesis that birds contribute to the introduction and re-emergence of EEEV to this enzootic focus.

A site near Tuskegee, Alabama was examined for vector activity of eastern equine encephalomyelitis (EEE) virus in 2001. More than 23,000 mosquitoes representing 8 genera and 34 species were collected during a 21-week period, and five species, Culiseta melanura, Aedes vexans, Coquillettidia perturbans, Culex erraticus, and Uranotaenia sapphirina, were examined for the presence of virus using a nested reverse transcriptase-polymerase chain reaction for EEE virus. Each species was infected at various times of the mosquito season (May-September) with different minimum infection rates (MIRs). Culiseta melanura had the highest MIR (20.2) and positive pools were detected from late May to mid-September. Aedes vexans had an MIR of 2.2 and was infected early in the season (June), while Cq. perturbans exhibited a much higher field infection rate (9.9) with all positive pools collected in August. Culiseta melanura is a likely endemic vector in central Alabama, while Ae. vexans and Cq. perturbans probably function as bridge vectors. Culex erraticus, the most common mosquito in the habitat (54% of total collections), had an MIR of 3.2, and was persistently infected from mid-June to mid-September. This is the first report of high rates of EEE virus infection in this species, a member of the tropical subgenus Melanoconion. Uranotaenia sapphirina, considered to feed on amphibians and possibly reptiles, had an MIR of 5.6, with positive pools spanning a four-month period. This suggests that species other than birds may serve as a reservoir for EEE in hardwood swamps in the Southeastern United States and elsewhere. The lengthy period of mosquito infection with EEE virus, coupled with the diverse habits of the vectors and their proximity to a population center, indicate the importance of monitoring EEE virus activity in the Mid-South.

In North America, the risk of mosquito-borne disease is thought to be shifting because of human development, with notable differences in disease risk between urban, rural, and natural ecosystems. In Eastern Ontario, I highlight differences in West Nile virus (WNV) and eastern equine encephalitis virus (EEEV) transmission risk to wildlife and exposure risk for Song Sparrows across urban-to-rural gradients. I found changes in mosquito host use between urban and rural environments, with potential mammal-based circulation of WNV occurring alongside a typical cycle between birds and mosquitoes. Additionally, I found significant differences in WNV and EEEV seroprevalence between Song Sparrows (Melospiza melodia) from urban, rural, and forested areas, with less WNV prevalence in forested areas and more EEEV prevalence in forested or urban habitats. I recommend continuing surveillance in mosquitoes and wildlife across urban and rural landscapes to better predict mosquito-borne disease outbreak potential.

West Nile Virus and Other Arboviral Diseases—United States, 2013

The emergence of the West Nile virus (WNV) in the northeastern United States has drawn emphasis to the need for expanded arbovirus surveillance in Connecticut. Although the state of Connecticut began a comprehensive mosquito-screening program in 1997, only since 1999 have there been efforts to determine the prevalence of arboviruses in bird populations in this state. Herein, we report on our results of an arbovirus survey of 1,704 bird brains. Included in this report are the first known isolations of eastern equine encephalitis virus (EEEV) from crows and data on the geographic and temporal distribution of 1,092 WNV isolations from crow species. Moreover, these nine isolations of EEEV identify regions of Connecticut where the virus is rarely found. With the exception of WNV and EEEV, no other arboviruses were isolated or detected. Taken together, these data illustrate the distribution of avian borne EEEV and WNV in 2000 and support the need for ongoing avian arbovirus surveillance in Connecticut.

Eastern equine encephalitis (EEE) virus perpetuates in an enzootic cycle involving ornithophilic mosquito vectors, principally Culiseta melanura (Coquillett) and avian amplification hosts. To better understand the role of Cs. melanura and Culiseta morsitans (Theobald) in the epizootiology of EEE virus, we collected blood-fed mosquitoes between 31 May and 15 October 2004 at two sites associated with an EEE virus focus in central New York and identified the source of vertebrate blood by nucleotide sequencing of polymerase chain reaction (PCR) products of the cytochrome b gene. Analysis of 484 Cs. melanura and 122 Cs. morsitans revealed that 94.2% and 86.9%, respectively, acquired blood solely from avian hosts. Blood meals derived exclusively from mammals were detected in 0.8% of Cs. melanura and 1.6% of Cs. morsitans. Individual mosquitoes containing mixed-blood meals from both avian and mammalian hosts were also detected in 5.0% of Cs. melanura and 11.5% of Cs. morsitans. Wood thrush constituted the most common vertebrate host for Cs. melanura (23.6%) and Cs. morsitans (30.9%), followed by American robin, song sparrow, ovenbird, red-eyed vireo, and common yellowthroat. Mammalian-derived blood meals were identified as white-tailed deer, horse, domestic cat, and eastern pipistrelle bat. There were three isolations of EEE virus from Cs. melanura and one from Cs. morsitans. These results suggest that wood thrush and a few other passerine birds may play key roles in supporting EEE virus transmission in the northeast and possibly throughout the geographic range of EEE in North America. The frequency of mammalian feedings also suggests that Cs. melanura and Cs. morsitans may play a role in the transmission of EEE virus to equines, in addition to maintaining enzootic transmission among avian hosts. We report the first isolation of arboviruses from mosquito vectors concomitant with the identifications of their blood meal sources.

Mosquito vectors of eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) in the USA reside within broad multi-species assemblages that vary in spatial and temporal composition, relative abundances and vector competence. These variations impact the risk of pathogen transmission and the operational management of these species by local public health vector control districts. However, most models of mosquito vector dynamics focus on single species and do not account for co-occurrence probabilities between mosquito species pairs across environmental gradients. In this investigation, we use for the first time conditional Markov Random Fields (CRF) to evaluate spatial co-occurrence patterns between host-seeking mosquito vectors of EEEV and WNV around sampling sites in Manatee County, Florida. Specifically, we aimed to: (i) quantify correlations between mosquito vector species and other mosquito species; (ii) quantify correlations between mosquito vectors and landscape and climate variables; and (iii) investigate whether the strength of correlations between species pairs are conditional on landscape or climate variables. We hypothesized that either mosquito species pairs co-occur in patterns driven by the landscape and/or climate variables, or these vector species pairs are unconditionally dependent on each other regardless of the environmental variables. Our results indicated that landscape and bioclimatic covariates did not substantially improve the overall model performance and that the log abundances of the majority of WNV and EEEV vector species were positively dependent on other vector and non-vector mosquito species, unconditionally. Only five individual mosquito vectors were weakly dependent on environmental variables with one exception, Culiseta melanura, the primary vector for EEEV, which showed a strong correlation with woody wetland, precipitation seasonality and average temperature of driest quarter. Our analyses showed that majority of the studied mosquito species’ abundance and distribution are insignificantly better predicted by the biotic correlations than by environmental variables. Additionally, these mosquito vector species may be habitat generalists, as indicated by the unconditional correlation matrices between species pairs, which could have confounded our analysis, but also indicated that the approach could be operationalized to leverage species co-occurrences as indicators of vector abundances in unsampled areas, or under scenarios where environmental variables are not informative.Graphical

BackgroundThe year 1971 was the first time in New York State (NYS) that Eastern equine encephalitis virus (EEEV) was identified in mosquitoes, in Culiseta melanura and Culiseta morsitans. At that time, state and county health departments began surveillance for EEEV in mosquitoes.MethodsFrom 1993 to 2012, county health departments continued voluntary participation with the state health department in mosquito and arbovirus surveillance. Adult female mosquitoes were trapped, identified, and pooled. Mosquito pools were tested for EEEV by Vero cell culture each of the twenty years. Beginning in 2000, mosquito extracts and cell culture supernatant were tested by reverse transcriptase-polymerase chain reaction (RT-PCR).ResultsDuring the years 1993 to 2012, EEEV was identified in: Culiseta melanura, Culiseta morsitans, Coquillettidia perturbans, Aedes canadensis (Ochlerotatus canadensis), Aedes vexans, Anopheles punctipennis, Anopheles quadrimaculatus, Psorophora ferox, Culex salinarius, and Culex pipiens-restuans group. EEEV was detected in 427 adult mosquito pools of 107,156 pools tested totaling 3.96 million mosquitoes. Detections of EEEV occurred in three geographical regions of NYS: Sullivan County, Suffolk County, and the contiguous counties of Madison, Oneida, Onondaga and Oswego. Detections of EEEV in mosquitoes occurred every year from 2003 to 2012, inclusive. EEEV was not detected in 1995, and 1998 to 2002, inclusive.ConclusionsThis was the first time in NYS that EEEV was detected in Cx. salinarius, Ps. ferox and An. punctipennis. The detection of EEEV in mosquitoes every year for 10 years was the longest time span since surveillance began in 1971. The calendar date of the earliest annual appearance of EEEV in mosquitoes did not change during surveillance spanning 42 years.